Endogenous Tagging of Ciliary Genes in Human RPE1 Cells for Live-Cell Imaging

Stefanie Kuhns; Alice Dupont Juhl; Zeinab Anvarian; Daniel Wüstner; Lotte B. Pedersen; Jens S. Andersen

doi:10.1007/978-1-0716-3507-0_9

Endogenous Tagging of Ciliary Genes in Human RPE1 Cells for Live-Cell Imaging

Stefanie Kuhns, Alice Dupont Juhl, Zeinab Anvarian, Daniel Wüstner, Lotte B. Pedersen, Jens S. Andersen^*

^*Kontaktforfatter

Københavns Universitet

Publikation: Kapitel i bog/rapport/konference-proceeding › Kapitel i bog › Forskning › peer review

Abstract

CRISPR-mediated endogenous tagging of genes provides unique possibilities to explore the function and dynamic subcellular localization of proteins in living cells. Here, we describe experimental strategies for endogenous PCR-tagging of ciliary genes in human RPE1 cells and how image acquisition and analysis of the expressed fluorescently tagged proteins can be utilized to study the dynamic ciliary processes of intraflagellar transport and vesicular trafficking.

Originalsprog	Engelsk
Titel	Cilia : Methods and Protocols
Redaktører	Vito Mennella
Forlag	Humana Press
Publikationsdato	2024
Sider	147-166
ISBN (Trykt)	978-1-0716-3509-4, 978-1-0716-3506-3
ISBN (Elektronisk)	978-1-0716-3507-0
DOI	https://doi.org/10.1007/978-1-0716-3507-0_9
Status	Udgivet - 2024

Navn	Methods in Molecular Biology
Vol/bind	2725
ISSN	1064-3745

Bibliografisk note

Funding Information:
We thank Joel Pomerantz, Kazuhisa Nakayama, Michael Knop, Keith Joung, and Benjamin Kleinstiver for reagents and the Danish Molecular Biomedical Imaging Center, University of Southern Denmark, for use of imaging equipment, supported by Novo Nordisk Foundation (NNF18SA0032928). This work was supported by grants from Independent Research Fund Denmark (grant 8021-00425B) to J.S.A. and Novo Nordisk Foundation (NNF18OC0053024, NNF15OC0016886 and NNF14OC0011535), The Danish Cancer Society (R146-A9590) and Hartmann Foundation (A31662) to L.B.P.

Dokumenter og links

10.1007/978-1-0716-3507-0_9

SDU link

Tjek adgangen til materialet i Syddansk Universitetsbiblioteks søgemaskine

1 Afsluttet

Novo Nordisk fonden - SDU bioimaging Infrastructure
Brewer, J. (Overordnet koordinator)
01/01/2019 → 31/12/2023
Projekter: Projekt › Forskning

Citationsformater

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abstract = "CRISPR-mediated endogenous tagging of genes provides unique possibilities to explore the function and dynamic subcellular localization of proteins in living cells. Here, we describe experimental strategies for endogenous PCR-tagging of ciliary genes in human RPE1 cells and how image acquisition and analysis of the expressed fluorescently tagged proteins can be utilized to study the dynamic ciliary processes of intraflagellar transport and vesicular trafficking.",

keywords = "Cas12a, Cas9, Cilia, CRISPR, Fluorescently tagged proteins, Image analysis, Intraflagellar transport, Live-cell imaging, Vesicle trafficking, Cilia/metabolism, Proteins/metabolism, Humans, Biological Transport, Protein Transport/genetics",

author = "Stefanie Kuhns and Juhl, \{Alice Dupont\} and Zeinab Anvarian and Daniel W{\"u}stner and Pedersen, \{Lotte B.\} and Andersen, \{Jens S.\}",

note = "Funding Information: We thank Joel Pomerantz, Kazuhisa Nakayama, Michael Knop, Keith Joung, and Benjamin Kleinstiver for reagents and the Danish Molecular Biomedical Imaging Center, University of Southern Denmark, for use of imaging equipment, supported by Novo Nordisk Foundation (NNF18SA0032928). This work was supported by grants from Independent Research Fund Denmark (grant 8021-00425B) to J.S.A. and Novo Nordisk Foundation (NNF18OC0053024, NNF15OC0016886 and NNF14OC0011535), The Danish Cancer Society (R146-A9590) and Hartmann Foundation (A31662) to L.B.P. ",

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language = "English",

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Endogenous Tagging of Ciliary Genes in Human RPE1 Cells for Live-Cell Imaging. / Kuhns, Stefanie; Juhl, Alice Dupont; Anvarian, Zeinab et al.
Cilia: Methods and Protocols. red. / Vito Mennella. Humana Press, 2024. s. 147-166 (Methods in Molecular Biology, Bind 2725).

Publikation: Kapitel i bog/rapport/konference-proceeding › Kapitel i bog › Forskning › peer review

TY - CHAP

T1 - Endogenous Tagging of Ciliary Genes in Human RPE1 Cells for Live-Cell Imaging

AU - Kuhns, Stefanie

AU - Juhl, Alice Dupont

AU - Anvarian, Zeinab

AU - Wüstner, Daniel

AU - Pedersen, Lotte B.

AU - Andersen, Jens S.

N1 - Funding Information: We thank Joel Pomerantz, Kazuhisa Nakayama, Michael Knop, Keith Joung, and Benjamin Kleinstiver for reagents and the Danish Molecular Biomedical Imaging Center, University of Southern Denmark, for use of imaging equipment, supported by Novo Nordisk Foundation (NNF18SA0032928). This work was supported by grants from Independent Research Fund Denmark (grant 8021-00425B) to J.S.A. and Novo Nordisk Foundation (NNF18OC0053024, NNF15OC0016886 and NNF14OC0011535), The Danish Cancer Society (R146-A9590) and Hartmann Foundation (A31662) to L.B.P.

PY - 2024

Y1 - 2024

N2 - CRISPR-mediated endogenous tagging of genes provides unique possibilities to explore the function and dynamic subcellular localization of proteins in living cells. Here, we describe experimental strategies for endogenous PCR-tagging of ciliary genes in human RPE1 cells and how image acquisition and analysis of the expressed fluorescently tagged proteins can be utilized to study the dynamic ciliary processes of intraflagellar transport and vesicular trafficking.

AB - CRISPR-mediated endogenous tagging of genes provides unique possibilities to explore the function and dynamic subcellular localization of proteins in living cells. Here, we describe experimental strategies for endogenous PCR-tagging of ciliary genes in human RPE1 cells and how image acquisition and analysis of the expressed fluorescently tagged proteins can be utilized to study the dynamic ciliary processes of intraflagellar transport and vesicular trafficking.

KW - Cas12a

KW - Cas9

KW - Cilia

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KW - Fluorescently tagged proteins

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KW - Intraflagellar transport

KW - Live-cell imaging

KW - Vesicle trafficking

KW - Cilia/metabolism

KW - Proteins/metabolism

KW - Humans

KW - Biological Transport

KW - Protein Transport/genetics

U2 - 10.1007/978-1-0716-3507-0_9

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BT - Cilia

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PB - Humana Press

ER -

Endogenous Tagging of Ciliary Genes in Human RPE1 Cells for Live-Cell Imaging

Abstract

Bibliografisk note

Dokumenter og links

SDU link

Fingeraftryk

Relaterede projekter

Novo Nordisk fonden - SDU bioimaging Infrastructure

Citationsformater